Classic modalities for the treatment of diseases such as human cancers, autoimmune diseases, viral, bacterial, parasitic and fungal diseases include surgery, radiation chemotherapy, antibiotics or combination therapies. However, these therapies are not effective against a majority of these diseases. Alternate therapies for preventing or treating human diseases are greatly needed. In the past decade immunotherapy and gene therapy utilizing T-lymphocytes have emerged as new and promising methods for treating human disease, in particular human cancers.
The T cell receptor for antigen (TCR) is responsible for the recognition of antigen associated with the major histocompatibility complex (MHC). The TCR expressed on the surface of T cells is associated with an invariant structure, CD3. CD3 is assumed to be responsible for intracellular signaling following occupancy of the TCR by ligand.
The T cell receptor for antigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC). Complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T cell targets. Stimulation of the TCR/CD3 complex results in activation of the T cell and a consequent antigen-specific immune response. The TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.
Two forms of T cell receptor for antigen are expressed on the surface of T cells. These contain either α/β heterodimers or γ/δ heterodimers. T cells are capable of rearranging the genes that encode the α, β, γ and δ chains of the T cell receptor. T cell receptor gene rearrangements are analogous to those that produce functional immunoglobulins in B cells and the presence of multiple variable and joining regions in the genome allows the generation of T cell receptors with a diverse range of binding specificities. Each α/β or γ/δ heterodimer is expressed on the surface of the T cell in association with four invariant peptides. These are the γ, δ and ε subunits of the CD3 complex and the zeta chain. The CD3 γ, δ and ε polypeptides are encoded by three members of the immunoglobulin supergene family and are found in a cluster on human chromosome 11 or murine chromosome 9. The zeta chain gene is found separately from other TCR and CD3 genes on chromosome 1 in both the mouse and human. Murine T cells are able to generate a receptor-associated η chain through alternative splicing of the zeta mRNA transcript. The CD3 chains and the zeta subunit do not show variability, and are not involved directly in antigen recognition.
All the components of the T cell receptor are membrane proteins and consist of a leader sequence, externally-disposed N-terminal extracellular domains, a single membrane-spanning domain, and cytoplasmic tails. The α, β, γ and δ antigen-binding polypeptides are glycoproteins. The zeta chain has a relatively short ectodomain of only nine amino acids and a long cytoplasmic tail of approximately 110 amino acids. Most T cell receptor α/β heterodimers are covalently linked through disulphide bonds, but many γ δ receptors associate with one another non-covalently. The zeta chain quantitatively forms either disulphide-linked ζ-η heterodimers or zeta-zeta homodimers.
Another example of a type of receptor on cells of the immune system is the Fc receptor. The interaction of antibody—antigen complexes with cells of the immune system results in a wide array of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulating lymphocyte proliferation, phagocytosis and target cell lysis. All these interactions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. It is now well established that the diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of Fc receptors (FcRs).
FcRs are defined by their specificity for immunoglobulin isotypes. Fc receptors for IgG are referred to as FcγR, for IgE as FcεR, for IgA as FcαR, etc. Structurally distinct receptors are distinguished by a Roman numeral, based on historical precedent. Three groups of FcγRs, designated FcγRI, FcγRII, and FcγRIII are now recognized. Two groups of FcεR have been defined; these are referred to as FcγRI and FcεRII. Structurally related although distinct genes within a group are denoted by A, B, C. Finally, the protein subunit is given a Greek letter, such as FcγRIIIAα, FcγRIIIAγ.
Considerable progress has recently been made in defining the heterogeneity for IgG and IgE Fc receptors (FcγR, FcεR) through their molecular cloning. These studies make it apparent that Fc receptors share structurally related ligand binding domains, but differ in their transmembrane and intracellular domains which presumably mediate intracellular signaling. Thus, specific FcγRs on different cells mediate different cellular responses upon interaction with an immune complex. The structural analysis of the FcγRs and FcεRI has also revealed at least one common subunit among some of these receptors. This common subunit is the γ subunit, which is similar to the ζ or η chain of the TCR/CD3, and is involved in the signal transduction of the FcγRIII and FcεRI.
The low affinity receptor for IgG (FcγRIIIA), is composed of the ligand binding CD16α (FcγRIIIAα) polypeptide associated with the γ chain (FcγRIIIAγ). The CD16 polypeptide appears as membrane anchored form in polymorphonuclear cells and as transmembrane form (CD16TM) in NK. The FcγRIIIA serves as a triggering molecule for NK cells.
Another type of immune cell receptor is the IL-2 receptor. This receptor is composed of three chains, the α chain (p55), the β chain (p75) and the γ chain. When stimulated by IL-2, lymphocytes undergo proliferation and activation.
Antigen-specific effector lymphocytes, such as tumor specific T cells (Tc), are very rare, individual-specific, limited in their recognition spectrum and difficult to obtain against most malignancies. Antibodies, on the other hand, are readily obtainable, more easily derived, have wider spectrum and are not individual-specific. The major problem of applying specific antibodies for cancer immunotherapy lies in the inability of sufficient amounts of monoclonal antibodies (mAb) to reach large areas within solid tumors. In practice, many clinical attempts to recruit the humoral or cellular arms of the immune system for passive anti-tumor immunotherapy have not fulfilled expectations. While it has been possible to obtain anti-tumor antibodies, their therapeutic use has been limited so far to blood-borne tumors [Lowder, J. N. et al. Cancer Surv. 4:359-375 (1985); Waldmann, T. A. Science 252:1657-1662 (1991)] primarily because solid tumors are inaccessible to sufficient amounts of antibodies [Jain, R. K. J. Natl. Cancer Inst. 81:64-66 (1989)]. The use of effector lymphocytes in adoptive immunotherapy, although effective in selected solid tumors, suffers on the other hand, from a lack of specificity (such as in the case of lymphokine-activated killer cells (LAK cells) [Mule, J. J. et al. Science 225:1487-1489 (1984)] which are mainly NK cells) or from the difficulty in recruiting tumor-infiltrating lymphocytes (TILs) and expanding such specific T cells for most malignancies [Rosenberg, S. A. et al. Science 233:1318-1321 (1986)]. Yet, the observations that TILs can be obtained in melanoma and renal cell carcinoma tumors, that they can be effective in selected patients and that foreign genes can function in these cells [Rosenberg, S. A. J. Clin. Oncol. 10:180-199 (1992)] demonstrate the therapeutic potential embodied in these cells.
A strategy which has been developed (European Published Patent Application No. 0340793) allows one to combine the advantage of the antibody's specificity with the homing, tissue penetration, cytokine production and target-cell destruction of T lymphocytes and to extend, by ex vivo genetic manipulations, the spectrum of anti-tumor specificity of T cells. Chimeric T cell receptor (cTCR) genes composed of the variable region domain (Fv) of an antibody molecule and the constant region domain of the antigen-binding TCR chains, i.e., the α/β or γ/δ chains have been expressed in T cells and found to be functionally active. Adoptive immunotherapies using tumor infiltrating lymphocytes and IL-2 have been developed for some cancers. These therapies have resulted in significant long-term responses in some patients with melanoma.
In an effort to broaden the applicability of adoptive immunotherapy to common cancers, such as, for example, ovarian, breast and colon cancer treatments that redirect the immune reactivity of lymphocytes to antigens recognized by monoclonal antibodies have been developed. To do this, retroviral vectors that encode chimeric receptor genes consisting of the variable regions of a monoclonal antibody joined to the transmembraneous and cytoplasmic domains of a T-cell receptor (TCR) signaling chain have been utilized. Using this approach, the safety of the administration of these chimeric receptor-transduced lymphocytes has been demonstrated. However, a need for improving the effectiveness of the chimeric receptor-transduced lymphocytes exists.
Thus, one object of the present invention is to produce an activated chimeric receptor-transduced lymphocyte capable of binding to and obliterating cancer cells.